RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation


In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor α (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.

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Figure 1: RNF4 preferentially binds poly-SUMO-2 chains.
Figure 2: Four SIMs in RNF4 are required to bind SUMO polymers.
Figure 3: RNF4 preferentially ubiquitinates poly-SUMO-2 chains.
Figure 4: RNF4 knockdown causes accumulation of SUMO-conjugated PML in ND10 nuclear domains.
Figure 5: Proteasome inhibition causes accumulation of PML, SUMO-1 and ubiquitin covalently associated with SUMO-2 in HeLa cells.
Figure 6: RNF4 ubiquitinates PML only when conjugated by SUMO-2.
Figure 7: Arsenic-mediated degradation of PML is RNF4-dependent.

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This work was supported by Cancer Research UK and the RUBICON EU Network of Excellence. A. P was supported by a Wellcome Trust Studentship. We would like to thank Hugues de The for helpful discussions and providing the chicken anti-PML antibody, and Douglas Lamont, manager of the Fingerprints Proteomics Facility, University of Dundee, for generating the Orbitrap mass spectrometry data. The provision of critical reagents by Roel van Driel and Dan Bailey is gratefully acknowledged.

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M. H. T. carried out the biochemical and proteomic analyses and participated in the writing of the manuscript; M. C. G. carried out the in vivo analysis of PML degradation; L. S. generated and assayed expressed proteins and mutants of RNF4; A. P. carried out ubiquitin site-mapping on poly-SUMO chains modified in vitro; N. H. was involved in initial immunofluorescence microscopy studies; E. J. G. established conditions for siRNA depletion of RNF4; J. J. P. generated expression constructs and antibodies to RNF4; R. T. H. conceived the project and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Ronald T. Hay.

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The authors declare no competing financial interests.

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Tatham, M., Geoffroy, MC., Shen, L. et al. RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat Cell Biol 10, 538–546 (2008). https://doi.org/10.1038/ncb1716

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